Many organic chemicals are manufactured using fermentation, including citric acid, acetic acid and lactic acid. Fermentation is also used to produce other products such as vitamins, amino acids, biofuels such as ethanol, enzymes or recombinant proteins. Demain, A. L and S. Sanchez, “Microbial Synthesis of Primary Metabolites: Current Advances and Future Prospects,” in El-Mansi, E., C. Bryce, A. Demain and A. Allman (eds.), “Fermentation Microbiology and Biotechnology (2d ed). CRC Press, Boca Raton, Fla. (2007).
The cost of fermentation substrates is one of the largest costs associated with industrial fermentation. Vasileva-Tonkova, E., M. Nustorova and A. Gushterova, “New Protein Hydrolysates from Collagen Wastes Used as Peptone for Bacterial Growth. Current Microbiology, 54:54-57 (2007); Ezeji, T., N. Qureshi, H. Blaschek, “Butanol Production from Agricultural Residues: Impact of Degradation Products on Clostridium beijerinckii Growth and Butanol Fermentation.” Wiley Interscience, (2007). Many organic chemicals which are now produced using petroleum-based raw materials could potentially be produced from renewable biobased raw materials through the use of industrial fermentation. In many cases, the high costs of existing fermentation substrates is a primary factor limiting the biobased production of these chemicals. Saha, B., “A low-cost medium for mannitol production by Lactobacillus intermedius NRRL B-3693. Appl. Microbiol. Biotechnol. 72:676-680 (2006). Among the many chemicals which could potentially be produced using fermentation are succinic acid, malic acid, glutamic acid, aspartic acid and 3-hydroxypropionic acid. Werpy, T. and G. Peterson (eds.), “Top Value Added Chemicals from Biomass.” National Renewable Energy Laboratory and Pacific Northwest National Laboratory (August 2004).
The dramatic expansion in recent years of the biofuels and biobased products industries has created a new urgency to develop improved and less expensive fermentation substrates. In some cases, it is possible to use inexpensive sources of sugar such as glucose or sucrose and inexpensive sources of protein such as soy meal or corn steep liquor for fermentation processes. However, many organic chemicals cannot presently be effectively produced using these inexpensive products and require more complex and expensive substrates such as yeast extract or peptones. However, it is also possible that supplementation of inexpensive sugar and protein sources with inexpensive new substrates might make it possible to produce organic chemicals which cannot now be produced through fermentation except with existing complex substrates.
Lactic acid is a chemical of particular interest. Long used in the food, chemical and pharmaceutical industries, lactic acid has attracted increasing attention in recent years due in part to its application in the manufacture of biodegradable polyacetate polymers, which are an alternative to non-biodegradable plastics.
Lactic acid can be industrially produced through fermentation, often using a lactic acid bacteria species such as the Lactobacillus sp. Kious, J., “Lactobacillus and Lactic Acid Production.” National Renewable Energy Laboratory (2000). However, the costs of fermentation substrates are a major factor in production economics. Yeast extracts and peptones are important nitrogen sources for lactic acid production, with yeast extract typically accounting for approximately 38% of production medium costs. Altaf, M., M. Venkateshwar, M. Srijana and G. Reddy, “An economic approach for L-(+) lactic acid fermentation by Lactobacillus amylophilus GV6 using inexpensive carbon and nitrogen sources.” J. of Appl. Microbiol. 103:372-380 (2007).
Another critical need in the industrial fermentation area relates to biofuels, i.e., fuels from biobased products. The bacterial species Escherichia coli (E. coli) is widely used in the production of ethanol, including cellulosic ethanol. E. coli can also be used to produce butanol and other ethanol alternatives. Thus substrates which can stimulate the growth of E. coli might have particular economic value.
Many current commercial microbial growth media are based on animal sources, such as milk or meat. This can pose risks of disease transmission, particularly if the fermentation product is intended for human consumption. In addition, use of animal-derived media is inappropriate if the fermentation products intended for vegetarians. Therefore, there is an interest in new plant-based growth media to replace existing milk or meat-based products.
In addition to industrial fermentations, fermentation substrates can also be used to as “prebiotics”, i.e., substances used to stimulate the growth of “probiotic” microorganisms. A probiotic is a live microbial food supplement that beneficially affects a host animal by improving its intestinal microbial balance, particularly the environment of the gastrointestinal tract. Probiotics are consumed either in food products or as dietary supplements. Postulated health advantages associated with probiotic intake are the 1) alleviation of symptoms of lactose malabsorption; 2) increase in natural resistance to infectious diseases of the intestinal tract; 3) suppression of cancer; 4) reduction in serum cholesterol concentrations; 5) improved digestion; and 6) stimulation of gastrointestinal immunity.
Prebiotics are used to stimulate the growth of probiotics. A prebiotic is a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth, activity, or both of one or a limited number of bacterial species already resident in the colon.
Yeh et al. found that a leaf hydrolysate prepared using the Chinese wolfberry, a medicinal herb widely used in Asia as a tea, somewhat stimulated the growth of the probiotic bacterial species Pediococcus acidilactici, compared to a control containing the bacterial growth medium MRS (de Man, Ragosa and Sharpe). Yeh, Y-C et al., “Effects of Chinese wolfberry (Lycium chinense P. Mill.) leaf hydrolysates on the growth of Pediococcus acidilactici.” Bioresource Technology 99: 1383-1393 (2008). The authors reported an approximately 2.5-fold increase in growth using a 20% treatment of the hydrolysate in an MRS medium compared to an MRS control. The authors reported P. acidilactici concentrations of about 5.5×109 CFU/ml after 24 hours. Based on this result, the authors suggested that wolfberry leaf hydrolysate may have potential for promoting the growth of probiotic bacteria. The authors did not evaluate or discuss the potential of wolfberry leaf hydrolysate to promote the growth of organic acids, nor did they raise the possibility of its suitability as a substrate in industrial fermentations.
Martin et al. found that hydrolysates prepared from tobacco stalks showed potential as raw material for conversion to cellulosic ethanol by fermentation with baker's yeast. Martin, C., et al., “Preparation of hydrolysates from tobacco stalks and ethanolic fermentation by Saccharomyces cerevisiae,” World J. Microbiol & Biotechnol. 18: 857-862 (2002). These researchers, however, only discussed conversion of the stalk matter, and did not discuss hydrolysis or other uses of the leaf. Furthermore, their research was limited to the suitability of tobacco stalks as a raw material for conversion to ethanol. They did not discuss the potential of tobacco stalks to serve as a substrate in the production of industrial chemicals.
Pandolfino reported that it was possible to produce ethanol from tobacco plants using a recombinant low-nicotine tobacco variety. U.S. Patent Application 20020197688 (Dec. 26, 2002). Pandolfino explained that ethanol could be produced by fermenting the plant in a fermentation vessel for a time sufficient to produce ethanol therefrom; and then collecting the ethanol from the fermentation vessel. Unlike Pandolfino, the claimed invention does not require a recombinant tobacco variety. Furthermore, the claimed invention does not relate to a method for producing ethanol but rather to produce a microbial growth promoter.
Levie et al. disclosed a method for producing a hydrolysate from lignocellulosic materials which involved fiberizing the materials and then separating them into two different portions. U.S. Patent Application 20080227161 (Sep. 18, 2008). The first portion would then be treated to deactivate lignin, and then the two portions would then be recombined prior to hydrolysis of the lignocellulosic materials. The resulting hydrolysate could then be fermented into ethanol. Levie's method required separation of the fiberized materials into two portions, with at least the first portion containing lignin, and then treating the first portion prior to recombination of the two portions and then hydrolyzing the combined materials with enzymes. Their objective was to use the hydrolysate to produce ethanol, as opposed to using it to stimulate microbial growth. In contrast to Levie, the claimed method does not relate to a microbial growth promoter and it does not require separation of the biomass into multiple portions.
Fichtali et al. taught a method for developing a stable composition by emulsifying a biomass hydrolysate which contained a desired nutrient. U.S. Patent Application 20060286205 (Dec. 21, 2006). The resulting emulsion product could be incorporated, into, or used as, nutritional products, cosmetic products or pharmaceutical products. The invention particularly related to stable compositions comprising at least one long chain polyunsaturated fatty acid. In contrast to Fichtali, the claimed method does not involve emulsification of the biomass hydrolysate. Also unlike Fichtali, the claim invention relates to a microbial growth promoter which is not one of the products produced through Fichtali's method.
There remains a need in the art for low cost materials that can be used to grow microorganisms. This need and others are met by the present invention.